Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell

ABSTRACT We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo [3,4-d]pyrimidine-4-one , the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC 50 ϭ 55 nM) and murine (IC 50 ϭ 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca 2ϩ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity

Synthesis and alkyne coordination chemistry of thiacycloalkynes

The reactions of 1,4-dichlorobut-2-yne with HS(CH2)(n)SH (n = 2-5) or HS(CH2CH2S)(n)H(n = 2-3) or HSCH2CH(OH)CH2SH under high dilution conditions in industrial methylated spirits using KOH as the deprotonating agent produce thiacycloalkynes in remarkably high yields when the 1 : 1 product is a nine-, ten- or 11-membered ring. 1,5-dithiacyclonon-7-yne, 1,5-dithiacyclonon-7-yne-3-ol, 1,6-dithiacyclodec-8-yne, 1,7-dithiacycloundec-9-yne, 1,4,7-trithiacycloundec-9-yne and 1,7-dithia-4-oxa-cycloundec-9-yne are pre pared in yields of 75%, 81%, 79%, 85%, 100% and 95% respectively. The X-ray crystal structures of 1,4-dithiacyclooct-6-yne and 1,6-dithiacyclodec-8-yne have been determined. Selective coordination of the alkyne functionality is demonstrated by the reaction of 1,4,7,10-tetrathiacyclotetradec-12-yne with [Co-2(CO)(8)] to afford a monoalkyne hexacarbonyldicobalt complex and by the reaction of 1,4,7-trithiacycloundec-9-yne with [Mo(CO)(2)(S(2)CNMe(2))(2)] to afford a bisalkynebis(dithiocarbamate)molybdenum complex. A base-catalysed rearrangement of 1,4,7-trithiacycloundec-9-yne affords 2-enyl-1,4,7-trithiacyclonon-2-ene which is an effective ligand for Ag+

Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc–protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy